Extracellular vesicles (EVs) have emerged as key regulators of immune function across multiple diseases. Severe burn injury is a devastating trauma with significant immune dysfunction that results in an ∼12% mortality rate due to sepsis‐induced organ failure, pneumonia, and other infections. Severe burn causes a biphasic immune response: an early (0–72 h) hyper‐inflammatory state, with release of damage‐associated molecular pattern molecules, such as high‐mobility group protein 1 (HMGB1), and proinflammatory cytokines (e.g., IL‐1β), followed by an immunosuppressive state (1–2+ wk post injury), associated with increased susceptibility to life‐threatening infections. We have reported that early after severe burn injury HMGB1 and IL‐1β are enriched in plasma EVs. Here we tested the impact of EVs isolated after burn injury on phenotypic and functional consequences in vivo and in vitro using adoptive transfers of EV. EVs isolated early from mice that underwent a 20% total body surface area burn injury (burn EVs) caused similar hallmark cytokine responses in naïve mice to those seen in burned mice. Burn EVs transferred to RAW264.7 macrophages caused similar functional (i.e., cytokine secretion) and immune gene expression changes seen with their associated phase of post‐burn immune dysfunction. Burn EVs isolated early (24 h) induced MCP‐1, IL‐12p70, and IFNγ, whereas EVs isolated later blunted RAW proinflammatory responses to bacterial endotoxin (LPS). We also describe significantly increased HMGB1 cargo in burn EVs purified days 1 to 7 after injury. Thus, burn EVs cause immune outcomes in naïve mice and macrophages similar to findings after severe burn injury, suggesting EVs promote post‐burn immune dysfunction.
Severe burn injury is a devastating form of trauma that results in persistent immune dysfunction with associated morbidity and mortality. The underlying drivers of this immune dysfunction remain elusive, and there are no prognostic markers to identify at-risk patients. Extracellular vesicles (EVs) are emerging as drivers of immune dysfunction as well as biomarkers. We investigated if EVs after burn injury promote macrophage activation and assessed if EV contents can predict length of hospital stay. EVs isolated early from mice that received a 20% total body surface area (TBSA) burn promoted proinflammatory responses in cultured splenic macrophages. Unbiased LC-MS/MS proteomic analysis of early EVs (<72 h post-injury) from mice and humans showed some similarities including enrichment of acute phase response proteins such as CRP and SAA1. Semi-unbiased assessment of early human burn patient EVs found alterations consistent with increased proinflammatory signaling and loss of inhibition of CRP expression. In a sample of 50 patients with large burn injury, EV SAA1 and CRP were correlated with TBSA injury in both sexes and were correlated with length of hospital stay in women. These findings suggest that EVs are drivers of immune responses after burn injury and their content may predict hospital course.
BACKGROUND:No methods exist to rapidly and accurately quantify the immune insult created by burn injuries. The development of a rapid, noninvasive clinical biomarker assay that evaluates a burn patient's underlying immune dysfunction and predicts clinical outcomes could transform burn care. We aimed to determine a set of peripheral biomarkers that correlates with clinical outcomes of burn patients. METHODS:This prospective observational study enrolled two patient cohorts within a single burn center into an institutionally approved institutional review board study. Blood draws were performed <48 hours after injury. Initial unbiased immune gene expression analysis compared 23 burn patients and 6 healthy controls using multiplex immune gene expression analysis of RNA from peripheral blood mononuclear cells. We then performed confirmatory outcomes analysis in 109 burn patients and 19 healthy controls using a targeted rapid quantitative polymerase chain reaction. Findings were validated and modeled associations with clinical outcomes using a regression model. RESULTS:A total of 149 genes with a significant difference in expression from burn patients compared with controls were identified. Pathway analysis identified pathways related to interleukin (IL)-10 and inducible nitric oxide synthase signaling to have significant z scores. quantitative polymerase chain reaction analysis of IL-10, IL-12, arginase 1 (ARG1), and inducible nitric oxide synthase demonstrated that burn injury was associated with increased expression of ARG1 and IL-10, and decreased expression of nitric oxide synthase 2 (NOS2) and IL-12. Burn severity, acute lung injury, development of infection, failure of skin autograft, and mortality significantly correlated with expression of one or more of these genes. Ratios of IL-10/IL-12, ARG1/NOS2, and (ARG1-IL-10)/ (NOS2-IL-12) transcript levels further improved the correlation with outcomes. Using a multivariate regression model, adjusting for patient confounders demonstrated that (ARG1-IL-10)/(NOS2-IL-12) significantly correlated with burn severity and development of acute lung injury. CONCLUSION:We present a means to predict patient outcomes early after burn injury using peripheral blood, allowing early identification of underlying immune dysfunction.
Heparan sulfates (HSs) are widely expressed glycans in the animal kingdom. HS plays a role in regulating cell differentiation/proliferation, embryonic development, blood coagulation, inflammatory response, and viral infection. The amount of HS and its structural information are critically important for investigating the functions of HS in vivo. A sensitive and reliable quantitative technique for the analysis of HS from biological samples is under development. Here, we report a new labeling reagent for HS disaccharides analysis, 6-amino-N-(2-diethylamino)ethyl quinoline-2-carboamide (AMQC). The AMQC-conjugated disaccharides are analyzed by LC-MS/MS in positive mode, significantly improving the sensitivity. The use of AMQC coupled with authentic 13C-labeled HS disaccharide internal standards empowered us to determine the amount and the disaccharide composition of the HS on a single histological slide. We used this method to profile the levels of HS in the plasma/serum and tissues/organs to assist the disease prognosis in two animal models, including the acetaminophen (APAP)-induced acute liver injury mouse model and the burn injury mouse model. The method may uncover the roles of HS contributing to the diseases as well as provide a potential new set of biomarkers for disease diagnosis and prognosis.
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